The agglutinin-like sequence (
) gene family encodes cell-surface adhesins that interact with host and abiotic surfaces, promoting colonization by opportunistic fungal pathogens such as
. Studies of ...Als protein contribution to
adhesion would benefit from an accurate catalog of
gene sequences as well as insight into relative gene expression levels. Even in the genomics era, this information has been elusive: genome assemblies are often broken within
genes because of their extensive regions of highly conserved, repeated DNA sequences and because there are many similar
genes at different chromosomal locations. Here, we describe the benefit of long-read DNA sequencing technology to facilitate characterization of
loci. Thirteen
loci in
strain MYA-3404 were deduced from a genome assembly constructed from Illumina MiSeq and Oxford Nanopore MinION data. Although the MinION data were valuable, PCR amplification and Sanger sequencing of
loci were still required to complete and verify the gene sequences. Each predicted Als protein featured an N-terminal binding domain, a central domain of tandemly repeated sequences, and a C-terminal domain rich in Ser and Thr. The presence of a secretory signal peptide and consensus sequence for addition of a glycosylphosphatidylinositol (GPI) anchor was consistent with predicted protein localization to the cell surface. TaqMan assays were designed to recognize each
gene, as well as both alleles at the divergent
locus.
cells grown in five different
conditions showed differential expression of various
genes. To place the
data into a larger context, TaqMan assays were also designed and validated for analysis of
gene expression in
and
. These comparisons identified the subset of highly expressed
genes that were predicted to encode proteins with the most abundant cell-surface presence, prioritizing them for subsequent functional analysis. Data presented here provide a solid foundation for future experimentation to deduce
family contributions to
adhesion and pathogenesis.
The genera
Trichophyton
,
Microsporum
, and
Epidermophyton
include filamentous fungi that cause dermatophytosis, a superficial infection of the skin, stratum corneum, nail beds, and hair follicles. ...The ability of dermatophytes to adhere to these substrates and adapt to the host environment is essential for the establishment of infection. Several fungal enzymes and proteins participate in this adaptive response to the environment and to keratin degradation. Transcription factors such as PacC and Hfs1, as well as heat shock proteins, are involved in sensing and adapting to the acidic pH of the skin in the early stages of fungal–host interaction. During dermatophyte growth, with keratin as the sole carbon source, the extracellular pH shifts from acidic to alkaline. This creates an environment in which most of the known keratinolytic proteases exhibit optimal activity. These events culminate in the establishment and maintenance of the infection, which can be chronic or acute depending on the dermatophyte species. This review focuses on these and other molecular aspects of the dermatophyte–host interaction.
Although it is widely recognized that disruption of
ALS3
reduces the invasion of
Candida albicans
germ tubes into mammalian oral epithelial cells, the mechanism of this interaction was unexplored.
C. ...albicans
strains with structurally informed mutations to remove adhesive activity of the peptide-binding cavity (PBC) or aggregative activity mediated by the amyloid-forming region (AFR) were assessed for their ability to invade cultured human oropharyngeal epithelial cells. Initial assays utilized untreated fungal and epithelial cells. Subsequent work used epithelial cells treated with cytochalasin D and
C. albicans
cells treated with thimerosal to investigate invasion mediated by active penetration of germ tubes and epithelial cell induced endocytosis, respectively. Results demonstrated the importance of the PBC for the invasion process: loss of PBC function resulted in the same reduced-invasion phenotype as a
C. albicans
strain that did not produce Als3 on its surface. Invasion
via
active penetration was particularly compromised without PBC function. Loss of AFR function produced a wild-type phenotype in the untreated and thimerosal-treated invasion assays but increased invasion in cytochalasin D-treated epithelial cells. In previous work, reduced AFR-mediated Als3 aggregation increased
C. albicans
adhesion to cultured epithelial cell monolayers, presumably
via
increased PBC accessibility for ligand binding. Collectively, results presented here demonstrate that Als3 PBC-mediated adhesion is integral to its invasive function. These new data add to the mechanistic understanding of the role of Als3 in
C. albicans
invasion into mammalian oral epithelial cells.
Candida parapsilosis is an emergent opportunistic yeast among hospital settings that affects mainly neonates and immunocompromised patients. Its most remarkable virulence traits are the ability to ...adhere to prosthetic materials, as well as the formation of biofilm on abiotic surfaces. The Ndt80 transcription factor was identified as one of the regulators of biofilm formation by C. parapsilosis; however, its function in this process was not yet clarified. By knocking out NDT80 (CPAR2-213640) gene, or even just one single copy of the gene, we observed substantial alterations of virulence attributes, including morphogenetic changes, adhesion and biofilm growth profiles. Both ndt80Δ and ndt80ΔΔ mutants changed colony and cell morphologies from smooth, yeast-shaped to crepe and pseudohyphal elongated forms, exhibiting promoted adherence to polystyrene microspheres and notably, forming a higher amount of biofilm compared to wild-type strain. Interestingly, we identified transcription factors Ume6, Cph2, Cwh41, Ace2, Bcr1, protein kinase Mkc1 and adhesin Als7 to be under Ndt80 negative regulation, partially explaining the phenotypes displayed by the ndt80ΔΔ mutant. Furthermore, ndt80ΔΔ pseudohyphae adhered more rapidly and were more resistant to murine macrophage attack, becoming deleterious to such cells after phagocytosis. Unexpectedly, our findings provide the first evidence for a direct role of Ndt80 as a repressor of C. parapsilosis virulence attributes. This finding shows that C. parapsilosis Ndt80 functionally diverges from its homolog in the close related fungal pathogen C. albicans.
We aimed to evaluate the properties of a novel tissue conditioner containing a surface pre-reacted glass-ionomer (S-PRG) nanofiller. Tissue conditioners containing 0 (control), 2.5, 5, 10, 20, or 30 ...wt% S-PRG nanofiller or 10 or 20 wt% S-PRG microfiller were prepared. The S-PRG nanofillers and microfillers were observed using scanning electron microscopy. The ion release, acid buffering capacity, detail reproduction, consistency, Shore A0 hardness, surface roughness, and Candida albicans adhesion of the tissue conditioners were examined. The results indicated that the nanofiller particles were smaller and more homogeneous in size than the microfiller particles. In addition, Al, B, F, and Sr ions eluted from S-PRG were generally found to decrease after 1 day. Acid neutralization was confirmed in a concentration-dependent manner. The mechanical properties of tissue conditioners containing S-PRG nanofiller were clinically acceptable according to ISO standard 10139-1:2018, although the surface roughness increased with increasing filler content. Conditioners with 5–30 wt% nanofiller had a sublethal effect on C. albicans and reduced fungal adhesion in vitro. In summary, tissue conditioner containing at least 5 wt% S-PRG nanofiller can reduce C. albicans adhesion and has potential as an alternative soft lining material.
Full and partial restorations in dentistry must replicate the characteristics of the patient's natural teeth. Materials must have good mechanical properties and be non-toxic and biocompatible. ...Microbes, which can form biofilms, are constantly in contact with restorations. In this study, we investigate how well
adheres to a polymethyl methacrylate (PMMA) resin base with gold (Au) nanoparticles. We synthesized Au nanoparticles and characterized them. The average size of Au nanoparticles embedded in PMMA was 11 nm. The color difference ΔE between PMMA and PMMA/Au composites was 2.7 and was still esthetically acceptable to patients. PMMA/Au surfaces are smoother and more hydrophilic than pure PMMA surfaces, and the isoelectric point of both types of surfaces was 4.3. Above the isoelectric point, PMMA/Au surfaces are more negatively charged than PMMA surfaces. The added Au nanoparticles decreased the tensile strength, while the hardness did not change significantly. Adhesion measurements showed that PMMA surfaces modified with Au nanoparticles reduced the extent of microbial adhesion of
.
Ectophosphatases are surface membrane-bound proteins whose active sites face the extracellular medium. These enzymes have been reported in several microorganisms including a large number of medically ...relevant fungal species. An effective technique for identifying ectophosphatases is performing phosphatase activity assays using living intact cells. Biochemical characterization of these activities has shown their differential modulation by classical phosphatase inhibitors, divalent metals and pH range. The physiological roles of ectophosphatases are not well established; however, it has been suggested that these enzymes play important roles in nutrition, proliferation, differentiation, adhesion, virulence and infection. Adhesion to host cells is the first step in establishing a fungal infection and ectophosphatases may be one of the first parasite proteins that come into contact with the host cells. Several results indicate that ectophosphatase activities increase the capacity of fungi to adhere to the host cells. In this context, the present review provides an overview of recent discoveries related to the occurrence and possible roles of ectophosphatase activities in fungal cells.
This report details the efficacy of nitric oxide (NO)-releasing xerogel surfaces composed of N-(6-aminohexyl)aminopropyl trimethoxysilane (AHAP3) and isobutyltrimethoxysilane (BTMOS) against Candida ...albicans adhesion, viability, and biofilm formation. A parallel plate flow cell assay was used to examine the effect of NO on planktonic fungal cells. Nitric oxide fluxes as low as 14 pmol cm
−2
s
−1
were sufficient to reduce fungal adhesion by ∼49% over the controls after 90 min. By utilizing a fluorescence live/dead assay and replicate plating, NO flux was determined to reduce fungal viability in a dose-dependent manner. The formation of C. albicans biofilms on NO-releasing xerogel-coated silicon rubber (SiR) coupons was impeded when compared to control (non-NO-releasing) and bare SiR surfaces. The synergistic efficacy of NO and silver sulfadiazine against adhered fungal cells and biofilms is reported with increased killing and biofilm inhibition over NO alone.
Binding of yeast forms to human lung fibroblast cultures was analyzed, aiming to better understand the initial steps of
Paracoccidioides brasiliensis
infection in humans. A significant
P. ...brasiliensis
adhesion was observed either to fibroblasts or to their Triton X-100 insoluble fraction, which contains extracellular matrix and membrane microdomains enriched in glycosphingolipids. Since human lung fibroblasts express at cell-surface gangliosides, such as GM1, GM2, and GM3, the role of these glycosphingolipids on
P. brasiliensis
adhesion was analyzed by different procedures. Anti-GM3 monoclonal antibody or cholera toxin subunit B (which binds specifically to GM1) reduced significantly fungal adhesion to fibroblast cells, by 35% and 33%, respectively. Direct binding of GM1 to yeast forms of
P. brasiliensis
was confirmed using cholera toxin subunit B conjugated to AlexaFluor
®
488. It was also demonstrated that
P. brasiliensis
binds to polystyrene plates coated with galactosylceramide, lactosylceramide, trihexosylceramide, GD3, GM1, GM3, and GD1a, suggesting that glycosphingolipids presenting residues of beta-galactose or neuraminic acid at non-reducing end may act as adhesion molecules for
P. brasiliensis
. Conversely, no binding was detected when plates were adsorbed with glycosphingolipids that contain terminal residue of beta-N-acetylgalactosamine, such as globoside (Gb4), GM2, and asialo-GM2. In human fibroblast (WI-38 cells), GM3 and GM1 are associated with membrane rafts, which remain insoluble after treatment with Triton X-100 at 4°C. Taken together, these results strongly suggest that lung fibroblast gangliosides, GM3 and GM1, are involved in binding and/or infection by
P. brasiliensis.
Maximum challenge exposure of Liposcelis bostrychophila to Beauveria bassiana, Paecilomyces fumosoroseus, Aspergillus parasiticus or Metarhizium anisopliae resulted in no more than 16% mortality. We ...investigated several of L. bostrychophila's cuticular lipids for possible contributions to its tolerance for entomopathogenic fungi. Saturated C14 and C16 fatty acids did not reduce the germination rates of B. bassiana or M. anisopliae conidia. Saturated C6 to C12 fatty acids that have not been identified in L. bostrychophila cuticular extracts significantly reduced germination, but the reduction was mitigated by the presence of stearamide. Cis-6-hexadecenal did not affect germination rates. Mycelial growth of either fungal species did not occur in the presence of caprylic acid, was reduced by the presence of lauric acid, and was not significantly affected by palmitic acid. Liposcelis bostrychophila is the only insect for which fatty acid amides have been identified as cuticular components. Stearamide, its major fatty amide, did not reduce germination of B. bassiana or M. anisopliae conidia or growth of their mycelia. Adhesion of conidia to stearamide preparations did not differ significantly from adhesion to the cuticle of L. bostrychophila. Pretreatment of a beetle known to be fungus-susceptible, larval Oryzaephilus surinamensis, with stearamide significantly decreased adhesion of B. bassiana or M. anisopliae conidia to their cuticles. This evidence indicates that cuticular fatty amides may contribute to L. bostrychophila's tolerance for entomopathogenic fungi by decreasing hydrophobicity and static charge, thereby reducing conidial adhesion.
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